1. Field of the Invention
The present invention is directed to the killing of neoplastic cells. More specifically, the present invention relates to the use of NADPH cytochrome P450 reductase (RED) to enhance cytochrome P450-based anti-cancer gene therapy.
2. Related Art
Traditional methods for cancer treatment rely on a combination of surgery, radiation, and cytotoxic chemotherapeutic drugs. Although the treatment of tumor cells with cytotoxic chemicals is well known in the art, presently, the therapeutic activity of many cytotoxic anti-cancer drugs is limited by a moderate therapeutic index associated with nonspecific toxicity toward normal host tissues, such as bone marrow, and the emergence of drug-resistant tumor cell sub-populations. One novel approach to enhancing the selectivity of cancer chemotherapeutics, and thereby reducing the toxicity of treatment, involves the application of gene therapy technologies to cancer treatment. See, Roth, J. A. and Cristiano, R. J., J. Natl. Cancer Inst. 89:21-39(1997); Rosenfeld, M. E. and Curiel, D. T., Curr. Opin. Oncol. 8:72-77 (1996).
In one such therapy known in the art, the phenotype of the target tumor cells is genetically altered to increase the tumors drug sensitivity and responsiveness. One promising strategy involves directly transferring a "chemosensitization" or "suicide" gene encoding a prodrug activation enzyme to malignant cells, in order to confer sensitivity to otherwise innocuous agents (Moolten, F. L., Cancer Gene Therapy 1:279-287 (1994); Freeman, S. M., et al., Semin. Oncol. 23:31-45 (1996); Deonarain, M. P., et al., Gene Therapy 2: 235-244 (1995)).
Several prodrug activation genes have been studied for application in cancer gene therapy. In one example, herpes simplex virus thymidine kinase (HSV-TK) in combination with the prodrug ganciclovir represents a prototypic prodrug/enzyme activation system known in the art with respect to its potential applications in cancer gene therapy. HSV-TK phosphorylates the prodrug ganciclovir and generates nucleoside analogs that induce DNA chain termination and cell death in actively dividing cells. Tumor cells transduced with HSV-TK acquire sensitivity to ganciclovir, a clinically proven agent originally designed for treatment of viral infections. Moolten, F. L. and Wells, J. M., J. Natl. Cancer Inst. 82:297-300 (1990); Ezzeddine, Z. D., et al., New Biol. 3:608-614 (1991).
In a second example, the bacterial gene cytosine deaminase (CD) is a prodrug/enzyme activation system that has been shown to sensitize tumor cells to the antifungal agent 5-fluorocytosine as a result of its transformation to 5-flurouracil, a known cancer chemotherapeutic agent (Mullen, C. A., et al., Proc. Natl. Acad. Sci. USA 89: 33-37 (1992); Huber, B. E., et al., Cancer Res. 53:4619-4626 (1993); Mullen, C. A., et al., Cancer Res. 54:1503-1506 (1994)). Recent studies using these drug susceptibility genes have yielded promising results. See, e.g., Caruso, M., et al., Proc. Natl. Acad. Sci. USA 90:7024-7028 (1993); Oldfield, E., et al., Hum. Gene Ther. 4: 39 (1993); Culver, K., Clin. Chem 40: 510 (1994); O'Malley, Jr., B. W., et al., Cancer Res. 56:1737-1741 (1996); Rainov, N. G., et al., Cancer Gene Therapy 3:99-106 (1996).
Several other prodrug-activating enzyme systems have also been investigated (T. A. Connors, Gene Ther. 2:702-709 (1995)). These include the bacterial enzyme carboxypeptidase G2, which does not have a mammalian homolog, and can be used to activate certain synthetic mustard prodrugs by cleavage of a glutamic acid moiety to release an active, cytotoxic mustard metabolite (Marais, R., et al., Cancer Res. 56: 4735-4742 (1996)), and E. coli nitro reductase, which activates the prodrug CB1954 and related mustard prodrug analogs (Drabek, D., et al., Gene Ther. 4:93-100 (1997); Green, N. K., et al., Cancer Gene Ther. 4:229-238 (1997)), some of which may be superior to CB1954 (Friedlos, F. et al., J Med Chem 40:1270-1275 (1997)). The principle underlying these approaches to prodrug activation gene therapy is that transduction of a tumor cell population with the foreign gene confers upon it a unique prodrug activation capacity, and hence a chemosensitivity which is absent from host cells that do not express the gene.
Current gene therapy technologies are limited by their inability to deliver prodrug activation or other therapeutic genes to a population of tumor cells with 100% efficiency. The effectiveness of this cancer gene therapy strategy can be greatly enhanced, however, by using drugs that exhibit a strong "bystander effect" (Pope, I. M., et al., Eur J Cancer 33:1005-1016 (1997)). Bystander cytotoxicity results when active drug metabolites diffuse or are otherwise transferred from their site of generation within a transduced tumor cell to a neighboring, naive tumor cell. Ideally, the bystander effect leads to significant tumor regression even when a minority of tumor cells is transduced with the prodrug activation gene (e.g., Chen, L., et al., Hum Gene Ther. 6:1467-1476 (1995); Freeman, S., et al., Cancer Res. 53:5274-5283 (1993)). Bystander cytotoxic responses may also be mediated through the immune system, following its stimulation by interleukins and other cytokines secreted by tumor cells undergoing apoptosis (Gagandeep, S., et al., Cancer Gene Ther. 3:83-88 (1996)).
Although the ganciclovir/HSV-TK and 5-fluorocytosine/CD systems have shown promise in preclinical studies, and clinical trials are underway (Eck, S. L., et al., Hum Gene Ther. 7:1465-1482 (1996); Link, C. J. et al., Hum Gene Ther. 7:1161-1179 (1996); Roth, J. A., and Cristiano, R. J., J Natl Cancer Inst. 89:21-39 (1997)), several limitations restrict their efficacy and limit their application to cancer chemotherapeutics. These include: (a) the non-mammalian nature of the HSV/TK and CD genes, whose gene products may elicit immune responses that interfere with prodrug activation; (b) their reliance on drugs which were initially developed as antiviral drugs (ganciclovir) or antifungal drugs (5-fluorocytosine) and whose cancer chemotherapeutic activity is uncertain; (c) the dependence of these gene therapy strategies on ongoing tumor cell DNA replication; and (d) the requirement, in the case of HSV-TK, for direct cell-cell contact to elicit an effective bystander cytotoxic response (Mesnil, M., et al., Proc. Natl. Acad. Sci. USA. 93: 1831-1835 (1996)). These considerations, together with the general requirement of combination chemotherapies to achieve effective, durable clinical responses, necessitates the development of alterative strategies to treat cancers using suicide gene-based (prodrug activation) gene therapy.
More recently, a drug activation/gene therapy strategy has been developed based on a cytochrome P450 gene ("CYP" or "P450") in combination with a cancer chemotherapeutic agent that is activated through a P450-catalyzed monoxygenase reaction (Chen, L. and Waxman, D. J., Cancer Research 55:581-589 (1995); Wei, M. X., et al., Hum. Gene Ther. 5:969-978 (1994); U.S. Pat. No. 5,688,773, issued Nov. 18, 1997). Unlike the prodrug activation strategies mentioned above, the P450-based drug activation strategy utilizes a mammalian drug activation gene (rather than a bacterially or virally derived gene), and also utilizes established chemotherapeutic drugs widely used in cancer therapy.
Many anti-cancer drugs are known to be oxygenated by cytochrome P450 enzymes to yield metabolites that are cytotoxic or cytostatic toward tumor cells. These include several commonly used cancer chemotherapeutic drugs, such as cyclophosphamide (CPA), its isomer ifosfamide (IFA), dacarbazine, procarbazine, thio-TEPA, etoposide, 2-aminoanthracene, 4-ipomeanol, and tamoxifen (LeBlanc, G. A. and Waxman, D. J., Drug Metab. Rev. 20:395-439 (1989); Ng, S. F. and Waxman D. J., Intl. J. Oncology 2:731-738 (1993); Goeptar, A. R., et al., Cancer Res. 54:2411-2418 (1994); van Maanen, J. M., et al., Cancer Res. 47:4658-4662 (1987); Dehal, S. S., et al., Cancer Res. 57:3402-3406 (1997); Rainov, N. G., et al., Human Gene Therapy 9:1261-1273 (1998)). Bioreductive metabolism that results in drug activation is also catalyzed by cytochrome P450 enzymes for a variety of anti-cancer drugs. Examples of such drugs include Adriamycin, mitomycin C, and tetramethylbenzoquinone (Goeptar, A. R., et al., Crit. Rev. Toxicol. 25:25-65 (1995); Goeptar, A. R., et al., Mol. Pharmacol. 44:1267-1277 (1993)).
CPA and IFA undergo bioactivation catalyzed by liver cytochrome P450 enzymes (Sladek, N. E., Pharmacol. Ther. 37:301-355 (1988)). Although IFA is an isomer of CPA, it is activated by a distinct subset of P450 enzymes, both in rodent models and in humans (Chang, T. K. H., et al., Cancer Res. 53:5629-5637 (1993); Weber, G. F. and Waxman, D. J., Biochem Pharmacol. 45:1685-1694 (1993)). The primary 4-hydroxy metabolite is formed at high levels in the liver and spontaneously decomposes, both in the circulation and within the target tumor cells, to yield acrolein and an electrophilic mustard, which exhibits the DNA crosslinking and cytotoxic effects associated with the parent drug. However, the systemic distribution of CPA and IFA and their alkylating metabolites inevitably results in several significant side effects, including cardiotoxicity, renal toxicity, marrow suppression, and neurotoxoxicity (Peters, W. P., et al., J. Clin. Oncol. 11:1132-1143 (1993); Ayash, L. J., et al., J. Clin. Oncol. 10:995-1000 (1992); Goren, M. P., et al., Lancet 2:1219-1220 (1986); Thigpen, T., Gynecol Oncol. 42:191-192 (1991)).
Some of these limitations can be overcome using a P450-based drug/enzyme activation system. In one example of this approach, tumor cells were rendered highly sensitive to CPA or IFA by transduction of CYP2B1, which encodes a liver P450 enzyme that exhibits a high rate of CPA and IFA activation (Clarke, L. and Waxman, D. J., Cancer Res. 49:2344-2350 (1989); Weber, G. F. and Waxman, D. J., Biochem. Pharmacol. 45:1685-1694 (1993)). This enhanced chemosensitivity has been demonstrated both in vitro and in studies using a subcutaneous rodent solid tumor model and human breast tumor grown in nude mice in vivo, and is strikingly effective in spite of the presence of a substantial liver-associated capacity for drug activation in these animals (Chen, L., et al., Cancer Res. 55:581-589 (1995); Chen, L., et al., Cancer Res. 56:1331-1340 (1996)). This P450-based approach also shows significant utility for gene therapy applications in the treatment of brain tumors (Wei, M. X., et al., Human Gene Ther. 5:969-978 (1994); Manome, Y., et al., Gene Therapy 3:513-520 (1996); Chase, M., et al., Nature Biotechnol. 16:444-448 (1998)).
Although the P450/drug activation system has shown great promise against several tumor types, further enhancement of the activity of this system is needed to achieve clinically effective, durable responses in cancer patients. This requirement is necessitated by two characteristics that are inherent to the P450 enzyme system: (1) P450 enzymes metabolize drugs and other foreign chemicals, including cancer chemotherapeutic drugs, at low rates, with a typical P450 turnover number (moles of metabolite formed/mole P450 enzyme) of only 10-30 per minute; and (2) P450 enzymes metabolize many chemotherapeutic drugs with high Km values, typically in the millimolar range. This compares to plasma drug concentrations that are only in the micromolar range for many chemotherapeutic drugs, including drugs such as CPA and IFA. Thus, current approaches to P450 gene therapy may result in intratumoral drug activation at a low absolute rate and under conditions that are not saturating with respect to drug substrate. Furthermore, since P450 is expressed at a very high level in liver tissue, only a very small fraction of the administered chemotherapeutic drug is metabolized via the tumor cell P450 gene product using the currently available methods for P450 gene therapy (Chen, L. and Waxman, D. J., Cancer Res. 55:581-589 (1995)).
Thus, in light of the foregoing, there is a need in the art for a method that will enhance the activity of a P450 gene product delivered to a tumor cell, in a manner that enhances the intratumoral drug activation reaction, in order to increase both the extent and selectivity of tumor cell destruction that occurs following treatment with a P450-activated chemotherapeutic agent. Moreover, in view of the radiation resistance and chemotherapeutic drug insensitivity that characterizes hypoxic tumor cells found in many human tumors (Brown, J. M. and Giaccia, A. J., Cancer Res. 58:1408-1416 (1998)), there is a need in the art for a method whereby a P450 gene product delivered to a tumor cell will kill hypoxic tumor cells, which may otherwise escape killing by classical treatment regimens.